1. Field of the Invention
The present invention is generally directed to methods of producing an increase in the enrichment and/or recovery of preferred forms of proteins. More particularly, the invention relates to methods for refolding recombinant antibody proteins.
2. Background of the Related Art
The advent of genetic engineering brought with it the promise of facile production of large quantities of biologically relevant polypeptides expressed in functional form in genetically-engineered organisms. In many instances, prokaryotes have been contemplated for use to achieve the expression of recombinant proteins. However, this promise has not been fully realized for a number of reasons. For example, in many instances where the polypeptide has been produced and retained in the cytoplasm of the host organism, inclusion bodies have resulted requiring denaturation and renaturation of the protein, frequently with only partial or little success. Many important target proteins are at best inefficiently expressed in soluble form in prokaryotic cells, due at least in part to the complexity of the protein folding process in vivo (Houry et al., Nature, 402: 147-154, 1999). Retrieval of the biologically active eukaryotic proteins from the inclusion bodies requires unfolding and refolding of the protein through the use of harsh conditions which include the use of chaotropic agents and reducing thiols. In other instances, the expressed protein or peptide is substantially degraded, not only leading to low yields but also generating complicated mixtures that are difficult to separate and purify.
Disulfide bond formation in proteins in vivo is a complex process, which is determined by the redox potential of the environment and specialized thiol-disulfide exchanging enzymes (Creighton, Methods Enzymol. 107, 305-329, 1984; Houee-Levin, Methods Enzymol. 353, 35-44, 2002; Ritz and Beckwith, Roles of thiol-redox pathways in bacteria, Annu. Rev. Microbiol. 55, 21-48, 2001.) The disulfides are formed in cells during or shortly after secretion of the nascent chains into the endoplasmic reticulum (Creighton, Methods Enzymol. 107, 305-329, 1984). Several conformational isoforms of the same protein, but with different disulfide structures, can be generated during recombinant protein production in mammalian cells due to the failing disulfide formation process, close proximity of three or more cysteine residues in the protein structure or surface exposure of unpaired cysteine residues.
In general, cysteine residues in proteins (including antibodies, IgG antibodies, IgG1 antibodies and the IgG1 antibody binding human IL-15) are either engaged in cysteine-cysteine disulfide bonds or sterically protected from the disulfide bond formation when they are a part of folded protein region. When a cysteine residue does not have a pair in protein structure and is not sterically protected by folding, it can form a disulfide bond with a free cysteine from solution (cysteinylation). The free cysteine residues are typically available in fermentation media together with other amino acids, building blocks of the proteins. The cysteinylation is undesirable posttranslational modification in pharmaceutical proteins, which may lead to a conformational isoform with undesirable properties, such as low binding, low biological activity and low stability. This invention provides method for removing the cysteinylation and increasing relative abundance of the desired conformational isoform without cysteinylation.
Unpaired cysteine residues in proteins can be subjected to cysteinylation, which can lead to significant changes in properties and function of the proteins. Cysteinylation of proteins was reported on proteins in vivo (Craescu et al., J. Biol. Chem. 261, 14710-14716, 1986; Dormann et al., J. Biol. Chem. 1993, 268, 16286-16292; Davis et al., Biochemistry 1996, 35, 2482-2488; Lim et al., Anal. Biochem. 2001, 295, 45-56, Bondarenko et al., Int. J. Mass Spectrom. Ion Processes 2002, 219, 671-680.) Modifications of cysteine residue modulated protein activity. For example, covalent binding of glutathione to hemoglobin increases the oxygen-binding properties of this protein (Craescu et al., J. Biol. Chem. 261, 14710-14716, 1986). In another example, liver type fatty acid-binding proteins (LABP) lost binding affinity after cysteinylation and glutathionylation (Dormann et al., J. Biol. Chem. 1993, 268, 16286-16292). HIV-1 protease activity was regulated through cysteinylation and glutathionylation (Davis et al., Biochemistry 1996, 35, 2482-2488). There are reports that there is a fraction of human antibodies in circulation that possesses an unpaired cysteine. For example, in one report it is shown that an immunoglobulin light chain of lambda type possesses a free cysteine in position 33, such that the light chain possesses a total of six cysteine residues (Buchwald et al., Can. J. Biochem. 1971, 49, 900-902). It was indicated that this free cysteine is a feature of a subgroup III of lambda light chains.
Although unpaired cysteines have been reported in IgG molecules there are no reported cases of cysteinylation of unpaired cysteins. Detection of cysteinylation can be analytically challenging and the failure to observe cysteinylation in earlier report could be due to the use of reduction in one of the steps in the analysis (reduction will eliminate cysteinylation). Cysteinylation when present in the CDR region can affect the biological activity as is seen in the case of 146B7, a fully human antibody directed against human IL-15. Removal of cysteinylation by refolding helps in minimizing heterogeneity hence improving product homogeneity. Removal of cysteinylation by refolding also increased product efficacy. There is a good chance that cysteinylation will be present on other IgG molecules containing one or more unpaired cysteines and removal of the cysteinylation could be the key for pharmaceutical viability of such products.
PCT Publication No. WO 02/68455 discloses a process for refolding a tumor necrosis factor receptor Fc fusion protein. The protein was bioengineered by fusing Fc region of IgG1 antibody and two tumor necrosis factor receptors (TNFr) and does not occur naturally. The document does not address proteins that have heterogeneous structures due to the presence of at least one free or unpaired cysteine, i.e., a cysteine that is not participating in a disulfide bond. Complex proteins bearing free cysteines are known to exist and at least some immunoglobulins are commercially relevant example of such proteins. In particular, it is noteworthy that WO 02/68455 provides no examples of processing of naturally occurring molecules such as immunoglobulins, nor does it discuss or address protein-folding problems of large complex proteins that contain free or unpaired cysteines.
In vitro folding of inclusion body proteins produced by microbial cells (E. coli) is well described in the literature and includes two steps. First, the inclusion body proteins are solubilized in a presence of high concentration of a chaotropic reagent and reducing reagent to break all disulfide bonds (Middelberg, A. P. Preparative protein refolding. Trends Biotechnol. 2002, 20, 437-443). For example, an inclusion body solubilization solution includes 6 M guanidine hydrochloride and 100 mM DTT in a review by Rudolph, R.; Lilie, H. In vitro folding of inclusion body proteins. FASEB J. 1996, 10, 49-56. The second step is protein folding in presence of a moderate concentration of guanidine hydrochloride (0.5-1.0 M) and a mild redox environment (Middelberg, A. P. Preparative protein refolding. Trends Biotechnol. 2002, 20, 437-443). This invention does not include the step of solubilization by protein complete denaturation and reduction of all disulfide bonds in a presence of the high concentrations of chaotropic and reducing agents. The invented method does not denature the protein or denatures it only and reduces/oxidizes (reshuffles) only a few disulfides. This invention is dealing with proteins produces in mammalian cells. The production by mammalian cells includes in vivo protein folding and disulfide formation, while microbial cells produce proteins as a high density, unfolded, non-soluble proteins agglomerates with mixed disulfides (inclusion bodies). Because the mammalian cells link most of the disulfide bonds correctly, there no need for complete protein denaturation and reduction of all disulfide bonds.
U.S. Pat. No. 4,766,205 recognizes that recombinant production of proteins is hampered by the formation of inappropriate intramolecular disulfide bonds that lead to “non-native” conformations of the recombinant protein that are “frozen” in that they cannot readily be converted to the native conformation. Such non-native products are at least partially biologically inactive. To address this issue, U.S. Pat. No. 4,766,205 discloses a process that involves exposure of the protein to a reductant, addition of an adduct forming disulfide compound, followed by addition of an oxidant with the temporally coordinated removal of the reductant. The detailed description of the invention indicates that proteins are subjected to solubilization by complete denaturation and reduction of disulfide bonds. The number of steps involved and the number of compounds required render this approach cumbersome. It is noteworthy that U.S. Pat. No. 4,766,205 provides no discussion on the use of the disclosed process for refolding mammalian produced proteins, and large complex proteins that are formed by intermolecular bonding, such as immunoglobulins.
The above discussion shows that there remains a substantial need and interest in developing systems for the efficient and economic production, purification and analysis of active large polypeptides where the desired polypeptide has been produced, for example through recombinant means, such that the produced polypeptide is provided in an active conformation or conveniently processed and renatured to a functional state. Additionally, despite the fact that there are techniques that have been extensively used in the analysis of low molecular weight proteins such as insulin, or low molecular weight digests of larger proteins, there remains a need for additional methods and techniques for producing sequence and detailed conformational information about larger proteins, in particular, proteins having more than one subunits that are formed by intermolecular interaction. The present invention is directed at addressing these needs.